Osmotic forces are not critical for Ca 2+ -induced secretion from permeabilized human neutrophils

Abstract

In order to examine the role of osmotic forces in degranulation, the effects of solutes and osmolality on granule secretion were explored using both FMLP-stimulated, intact neutrophils and Ca 2+ -stimulated, permeabilized cells. We employed a HEPES-based buffer system which was supplemented with: (a) permeant (KCl or NaCI) or impermeant (Na-isethionate or choline-CI) ions, or (b) permeant (urea) or impermeant (sucrose) uncharged solutes. Intact and permeabilized cells had significantly different solute requirements for degranulation. FMLP-stimulated release from intact cells was supported by NaCI or Na-isethionate > KCl > choline-Cl or sucrose > urea. In contrast, the rank order of Ca 2+ -stimulated release from permeabilized cells was choline-C > Na-isethionate, KCl, or NaCl > sucrose > urea. Hypo-osmotic conditions caused increased levels of background granule release from both intact and permeabilized neutrophils. However, hypo-osmolality inhibited both FMLP-stimulated degranulation from intact cells and Ca 2+ -induced release from permeabilized neutrophils. While hyperosmotic conditions inhibited stimulated release from intact cells, this inhibition was much less pronounced in permeabilized cells when the granules were directly exposed to these solutions. In fact, hyperosmotic sucrose greatly enhanced Ca 2+ -induced secretion. Although isolated specific and azurophil granules showed some lytic tendencies in hypo-osmotic buffers, the overall stability of the isolated granules did not indicate that swelling alone could effect degranulation. These results suggest that degranulation in permeabilized cells is neither due to nor driven by simple osmotic forces (under resting or stimulated conditions) and emphasize differences obtained by bathing both the granules and plasma membrane (as opposed to membranes alone) in various solutes.